Kinetochores are specialized protein complexes that are assembled on specific loci of chromosomes named centromeres. They mediate the interaction between chromosomes and the spindle microtubules, which separate chromosomes during mitosis. Aberrant function of kinetochore leads to aneuploidy, a major form of genetic instability implicated with a number of diseases, in particular, cancers. The goal of this application is to delineate the architecture of the kinetochore and to understand the mechanisms by which kinetochores mediate chromosome segregation in the fission yeast S.pombe. The fission yeast kinetochore is an excellent model for the study of "regional kinetochores" found in most eukaryotes, which bind to multiple microtubules. Questions unique to regional kinetochores can be addressed in molecular detail and the lessons learnt can be applied to human kinetochores. In contrast, the "point kinetochore" found in the budding yeast S.cerevisiae is the simple type and binds to one microtubule. We previously have made major progress in determining the biochemical composition of the kinetochore in the fission yeast. We have identified three major kinetochore complexes including thirty proteins. We have also determined that the centromeric foundation of a fission yeast kinetochore is composed of an array of well-positioned nucleosomes, among which three contain centromere-specific histone H3 variant Cenp-A (Cnp1p), corresponding to three microtubules bound to the kinetochore. Furthermore, our recent finding in Cnp1-nucleosome positions in the centromere indicates that Cnp1-nucleosomes occupy a subset of preferred but flexible positions. By determining the copy numbers of the representative components and comparing them to those in the budding yeast, we propose that a regional kinetochore in fission yeast is comprised of three functional units, each is architecturally similar to a point kinetochore. Importantly, fission yeast kinetochores do not possess sufficient copies of the Dam1 complex to assemble a 16-mer ring, a structure crucial for the Dam1's function as a coupler of chromosome movement and microtubule depolymerization in the budding yeast. In the next funding period, we will further investigate the molecular assembly of kinetochores in the fission yeast by testing a "repeat unit" model at the molecular level. The model postulates that the regional kinetochore in the fission yeast is comprised of multiple units, each unit is capable of binding to one MT. We will determine what comprises of the chromatin foundation of one unit and how multiple units are arranged to form a whole kinetochore (Aim 1). We will delineate the architectural features of the kinetochore unit and the comprehensive functional connections among the kinetochore components (Aim 2). We will also investigate the functions and the mechanism of fission yeast Dam1, which is also involved in coupling chromosome movement and microtubule depolymierization, but functions in the multimeric assemblies smaller than a 16- mer ring (Aim3). PUBLIC HEALTH RELEVANCE: This project studies how a kinetochore, a crucial component of the mitosis machinery, is assembled and functions in mediating chromosome segregation. This knowledge will have important therapeutic implications, since defects in chromosome segregation directly lead to abnormality in chromosome numbers - called aneuploidy. Aneuploidy is implicated in many human diseases, and in particular, is closely involved in tumorigenesis. This project will be conducted in a single cell organism, the fission yeast S.pombe, which is an excellent model for the study of the regional kinetochores, the type found in humans.